CN1065909C - Microorganisms for the production of tryptophan and process for the preparation thereof - Google Patents

Microorganisms for the production of tryptophan and process for the preparation thereof Download PDF

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CN1065909C
CN1065909C CN93117586A CN93117586A CN1065909C CN 1065909 C CN1065909 C CN 1065909C CN 93117586 A CN93117586 A CN 93117586A CN 93117586 A CN93117586 A CN 93117586A CN 1065909 C CN1065909 C CN 1065909C
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tryptophane
sera
bacterial strain
serine
plasmid
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冈特·维歇
沃尔弗来德·莱因菲尔德
基思·巴克曼
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Wacker Polymer Systems GmbH and Co KG
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Electrochemical Industry Co ltd International
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/09—Recombinant DNA-technology
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    • C12R2001/15—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
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    • Y10S435/843—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/848—Escherichia
    • Y10S435/849—Escherichia coli

Abstract

A tryptophan producing strain of microorganism is selected from E. coli and Corynebacteria and is tryptophan feedback resistant and serine feedback resistant. The serine feedback resistance is by a mutation in a serA allele, where the mutated serA allele codes for a protein which has a Ki value for serine between 0.1 mM and 50 mM. The tryptophan feedback resistance is by a trpE allele which codes for a protein which has a Ki value for tryptophan between 0.1 mM and 20 mM. A process for preparing this microorganism and a process for using this microorganism are disclosed.

Description

Produce microorganism of tryptophane and preparation method thereof
The present invention relates to microorganism that produces tryptophane and preparation method thereof.
Tryptophan metabolism only carries out (Somerville with a single creature route of synthesis in all microorganisms of being studied at present as everybody knows, R.L., Herrmann, K.M., 1983, amino acid, biosynthesizing and gene regulating (Aminoacids, Biosynthesisand Genetic Regulation), Addison-Wesley Publishing Company, USA:30l-322 and 351-378; Aida et al., 1986, the biological method that amino acid is produced, industrial microbiology progress (Biotechnology of aminoacidproduction, progress in industrial microbiology) Vol 24, ElsevierSciencepublishers, Amsterdam:188-206).The tryptophan metabolism approach and with serine metabolism get in touch and the gene of the main enzyme of encoding is listed among Fig. 1.
Known tryptophane production method is based on the trpE expression of gene of a sudden change, this genes encoding has the insensitive anthranilic acid of tryptophane (anthranilate) synthase, and based on other expression of gene of the trp operon on the suitable carrier that can duplicate automatically.Because the relative high copy number of these genes, the trp expression of gene is also many, and the amount of each enzyme in the corresponding tryptophan metabolism has also increased.This causes the excessive production of tryptophane.
Below for the example of some this production tryptophane methods, there are many microorganisms to be used for this process, as intestinal bacteria: EP0293207, US4,371,614, genus bacillus: US4,588,687, coryneform bacteria (Corynebacterium) and tyrothricin (Brevibacterium): EP0338474.In this production process, can exist unstable, carrier to lose or produce strain growth speed and wait problem slowly.
EP-A-O401735 (applicant: Kyowa Hakko Kogyo Co.) described the method for producing the L-tryptophane with the coryneform bacteria that contains recombinant plasmid or tyrothricin.In these plasmids, contain synthetic DAHP synthase, o-amino benzoyl acid synthase, indoles-3-glycerine-P synthase, the genetic information of tryptophan synthetase and phosphoglycerol dehydrogenase.Used the allelotrope of anti-feedback o-amino benzoyl acid synthase.
And then we also know and can introduce many serA by give producing bacterial strain, or serA, and B, C wild type gene cause tryptophan metabolism to go to regulate and control to increase the production of tryptophane.Therefore chemical abstracts CA111 (1989) 168688q and CA111 (1989) the 168689r Bacillus strain of having described whole wild type genes (serA, serB and serC) of the serA wild-type allele that is introduced in respectively on the plasmid and serine metabolism can superfluously be expressed them and be produced tryptophane.
EP-0149539 (applicant: disclosed Stauffer Chemical Company) by preventing that in cell Serine from degrading and improved the method for tryptophane output.(e. coli k12 mutant sda) has also been described simultaneously with this class bacterial strain and has been produced amino acid for serine deaminase, Serinedeaminase to have used destruction Serine degrading enzyme in this patent application.Such bacterial strain is described, the method for mass production tryptophane from anthranilic acid in the example VIII.Produce tryptophane with a kind of bacterial strain with complete serine deaminase that european patent application is mentioned and compare, the explanation that tryptophane output improves is the production of Serine or Serine biosynthesis ability speed limit tryptophane in the very high microorganism of amino acid precursor reserves.
The purpose of this invention is to provide the microorganism that can increase tryptophane output and providing makes this microorganism preparation become possible certain methods.
Purpose of the present invention can realize by microorganism strains.These bacterial strains are characterised in that having one goes (deregulated) tryptophan metabolism regulated and control and one to be removed the serine metabolism of regulating and control at least by an anti-feedback serA allelotrope.
Among the present invention, anti-feedback serA allelotrope site means the mutant of serA gene, its phosphoglycerol dehydrogenase that Serine is had susceptibility of encoding, but the Serine susceptibility of this enzyme will be lower than the Serine susceptibility of this bacterial strain corresponding wild type phosphoglycerol dehydrogenase.
The microorganism of at least one anti-feedback serA allelotrope and the tryptophan metabolism that goes to regulate and control combine the increase that meeting causes tryptophane output by the present invention.Surprisingly output is through comparing under identical culture condition with not having the allelic same microorganism of anti-feedback serA, and the former exceeds 2.6 times than the latter.
According to the present invention, making the raising of tryptophane output with bacterial strain of the present invention is that unanticipated arrives and wonderful, because anti-feedback serA allelotrope has shown only (the Tosa T of the effect on the Serine level in high cell, Pizer L.J., 1971, Journal ofBacteriology Vol.106:972-982; WinicovJ., Pizer L.J., 1974, Journal of Biological Chemistry Vol.249:1348-1355)., by the state of the art (for example EP-A-O149539), the bacterial strain with tryptophan metabolism of regulating and control has a very low Serine level.Why Here it is does not expect by the present invention is introduced the production that an anti-feedback serA allelotrope can improve tryptophane by remove bacterial strain to tryptophan metabolism.
Because tryptophan metabolism all is to be undertaken by pathways metabolism shown in Figure 1 in all known microorganisms, and it is known preparing on the engineering philosophy of bacterial strain of the present invention, and can be used for all microorganisms, so bacterial strain of the present invention can make from the microorganism of any hope.
That be fit to and preferably to be used for the bacterial strain that the present invention produces tryptophane be bacterium, particularly Gram-negative bacteria is to be more suitable for preferred as intestinal bacteria.
Bacterial strain of the present invention can obtain by partially or completely the tryptophan metabolism regulation and control of purpose tryptophane prototroph initial strain being abolished and introduced an anti-feedback serA allelotrope.
Bacterial strain of the present invention also can be similarly by the auxotrophic initial strain of tryptophane being replied its combination colour propylhomoserin ability, and remove regressive tryptophan metabolism, introduce anti-feedback serA allelotrope again and obtain.
The tryptophan metabolism of bacterial strain goes and can realize by many different this area prior arts.
A kind of possibility that tryptophan metabolism goes to regulate and control is to modify o-amino benzoyl acid synthase, the first step in all microorganisms of this enzyme catalysis in the specific biosynthetic pathway of tryptophane.Its activity is subjected to the inhibition of tryptophane, so its amount of relying on tryptophane regulates the metabolism logistics by the tryptophane biosynthetic pathway, and this enzyme is by the trpE genes encoding.
The susceptibility of the sudden change trpE gene pairs tryptophane of coding o-amino benzoyl acid synthase is lower than the susceptibility of corresponding wild type o-amino benzoyl acid synthase, therefore be also referred to as anti-feedback trpE allelotrope, it can and screen then by mutagenesis prototroph tryptophane initial strain and obtain.For reaching this point, relevant bacterial strain should be handled, lure its sudden change (MillerJ.H., 1972, Experiments in Molecular Genetics, Cold Spring HarborLaboratory, USA:113-185).
Processed strain culturing is on nutritional medium, and this substratum contains a kind of tryptophane antagonist at least, and its amount should be enough to suppress the growth of this bacterial strain.Suitable tryptophane antagonist such as 4-methyl-tryptophane, 5-methyl tryptophan, 6-methyl tryptophan, halogenation tryptophane, tryptazan (tryptazan), indoles and indole acrylic acid.
The tryptophane susceptibility of the o-amino benzoyl acid synthase of antagonism clone strain is measured.The tryptophane susceptibility of o-amino benzoyl acid synthase can adopt and anyly exist the method for measuring down enzymic activity to detect definite with tryptophane.For example, chorismic acid (chorismate) can react with glutamine in a suitable damping fluid, in this enzymatic reaction, and glutamine and tryptophane acting in conjunction (Bauerle R.et al., 1987, Methods in EnzymologyVol.142:366-386).From detect mixture, shift out equal portions, determine the amount of time per unit reaction after product anthranilic acid with high-efficient liquid phase chromatogram technique analysis in the kinetics mode.The amount of the product anthranilic acid that time per unit obtains is the direct mensuration of anthranilic acid synthase activity.In order to measure the susceptibility of o-amino benzoyl acid synthase to be measured, this method should exist and not deposit in both cases at tryptophane carries out.
Also available direct gene operation obtains the insensitive trpE allelotrope of tryptophane (Bauerle R.et al., 1987, Methods in Enzymology Vol.142:366-386).Various microorganisms are all existing to be described many variations on the o-amino benzoyl acid synthase aminoacid sequence and all will cause this enzyme to the reduction of tryptophane susceptibility (Salmonellas: CaliguiriMG. for example, Bauerle R., 1991, J.of Biol.Chem.Vol.266:8328-8335; Furbrevibacterium, corynebacterium:Matsui K.et al., 1987, J.Bact.Vol.169:5330-5332).
There are some currently known methodss to make that a specified point induced mutation has become possibility on dna fragmentation.These class methods are described in following public publication especially:
Sakar G., Sommerauer S.S., 1990, the site-directed mutagenesis that relies on polymerase chain reaction has been described among the Bio Techniques 8:404-407.
Ausubel F.M.et al., 1987, Currentprotocols in MolecularBiology, Greenepublishing Associates has described the method that relies on the M13 phage.
Smith M., 1985, Ann.Rev.Genet, 19:423-462 has described other method.
The dna fragmentation that contains wild-type trpE gene is recombinated with the early stage standard method of describing and is obtained recombinant DNA on the carrier.Use the above-mentioned site-directed mutagenesis method of mentioning will cause on the dna sequence dna one or more Nucleotide to be modified, the result is just corresponding with the aminoacid sequence to the insensitive o-amino benzoyl acid synthase of tryptophane by the aminoacid sequence of this genes encoding.The technology of this description can be used to any purpose trpE gene is introduced in one or more sudden changes, and this o-amino benzoyl acid synthase that will cause encoding has the insensitive aminoacid sequence of tryptophane.
In addition, the following properties of bacterial strain of the present invention is that hope obtains but is not absolute demand: the tryptophane aporepressor of defective (repressor protein), the decay control of the defective that the trp operon is expressed and the tryptophanase of defective, these features of bacterial strain of the present invention can be the most simply by selecting an initial strain that has had one or more suitable features to obtain.This preparation and selection can be by carrying out after the following system of selection combination of mentioning.
Tryptophane aporepressor (repressor protein) is the main albumen of regulating of in the tryptophane biosynthesizing.This protein and tryptophane check the expression of trp operon gene together as aporepressor (aporepressor), this albumen is by the trpR genes encoding.Can from the mutant that tryptophane antagonist (as 5-methyl tryptophan) is had resistance, filter out tryptophane repressor mutant.At J.Mol.Biol.44,1969,185-193 or Genetics 52,1965, existing example in the 1303-1316 document.
Except the control by the trpR proteins encoded was arranged, the trp operon had also participated in the decay regulation and control.DNA section before first gene of trp operon is responsible for regulation and control.The mutant of this section or deletant may cause regulation and control.This class mutant can screen from the mutant that tryptophane antagonist (as 5-methyl tryptophan) is had resistance.On the other hand, this class mutant, particularly deletant, the method for available site-directed mutagenesis locus specificity in the attenuation region of DNA imports this modification and obtains.Can recombinate the decay subarea of deactivation in the karyomit(e) of bacterial strain of the present invention to replace natural attenuator zone by the site-specific mutagenesis technology narrated.
Tryptophanase (tnaA) catalysis tryptophane is degraded into indoles, pyruvic acid (pyruvate) and ammonia.It is desirable to this kind of enzyme is non-activity in tryptophane production bacterial strain.The bacterial strain of disappearance this kind of enzyme can be by processing that bacterial strain is suddenlyd change, and searches out those and no longer can utilize tryptophane to obtain as the mutant of carbon nitrogen source.At document J.Bact.85,1965, give a detailed example among the 680-685, or may will cause the some specific mutant body of deactivation to introduce the tnaA gene with above-mentioned technology on an equal basis.
The very suitable further raising that causes tryptophane to be produced of other mutant of many initial strains.Therefore, tryptophane biosynthetic pathway not only.And general die aromatischen Aminosaeuren biosynthesizing way shikimic acid pathway, (shikimic acidpathway) preferably also is that regulation and control are insensitive.According to the present invention, why Here it is has the Arabic ketoheptose acid synthase (dehydroarabinoheptulosonate synthase) of dehydrogenation of regulation and control insensitivity and its tyrosine repressor albumen (tyrR) through sudden change or disappearance and the bacterial strain of deactivation preferably is used to prepare bacterial strain of the present invention as initial strain.Same phenylalanine and the weakened bacterial strain of tyrosine metabolism also are preferred.This has guaranteed that the precursor molecule branched acid all flows to the tryptophane direction.Such bacterial strain for example has pheA or/and sudden change on the tyrA gene or disappearance.
Remove regulation and control mass production tryptophane thereby many bacterial strains are known in a biosynthetic step of tryptophane or multistep or shikimic acid pathway.For example, Bacillus subtilus FermBP-4, FermP1483 (DE3123001), brevibacterium flavum (Brevibacterium flavum) ATCC 21427 (US3,849,251), corynebacterium glutamicum (corynebacteriumglutamicum) ATCC 21842-21851 (US3,594,279, US3,849,251), corpus luteum micrococci (Micrococcus luteus) ATCC 21102 (US3,385,762), intestinal bacteria ATCC 31743 (CA1182409), these bacterial strains are equally applicable to as initial strain and prepare production bacterial strain of the present invention.They show that all tryptophane production bacterial strain can obtain from the micropopulation of wide range according to the present invention.
Except the tryptophan metabolism that requires bacterial strain to have to regulate and control, the bacterial strain that also requires to be used to prepare bacterial strain of the present invention has the gene of a coding phosphoglycerol dehydrogenase at least, and the Serine susceptibility of this enzyme will be lower than the Serine susceptibility of the phosphoglycerol dehydrogenase of corresponding wild-type bacterial strain.
Phosphoglycerol dehydrogenase (PGD) is by the serA genes encoding.The sequence of known wild-type serA gene (Tobey K.L., Grant G.A., 1986, J.Bac.Vol, 261, No.26:1279-1283).With this wild-type of plasmid vector great expression serA gene prod known too (Schuller et al., 1989, J.Biol.Chem.Vol.264:2645-2648).
Genetic method preparation with classics resists feedback serA allelotrope by Tosa T, PizerL, and T., 1971, J.Bac.Vol.106, No.3:972-982 describes.In this embodiment, can screen the resistance of serine analogs Serine hydroxamic acid (hydroxamate) by mutant.These mutant do not identify in detail that in the document sudden change is also studied metabolic influence.
Anti-feedback serA allelotrope also can obtain by microorganism is suddenlyd change to induce.The mutagenic compound that are fit to are ultraviolet ray (UV) and some chemical mutagens, as ethylmethane sulfonate or N-methyl-N '-nitro-N-nitrosoguanidine.Dosage and action time are determined (MillerJ.H., 1972, Experiments inMolecular Genetics, Cold Spring Harbor Laboratory USA:113-143) by ordinary method to the mutagenic compound of selecting for use.
The micropopulation screening that mutagenic compound are handled has the clone strain of the serA gene of encoding serine insensitivity phosphoglycerol dehydrogenase then.For example, group who handles with mutagenic compound is inoculated into to contain on the Serine hydroxamate solid growth culture media that is enough to suppress non-resistance bacterial growth amount and cultivates.The Serine sensitivity testing of phosphoglycerol dehydrogenase is carried out in the resistance clone strain again to it.This method is by Tosa and pfizer, and 1971, J.Bact, 100, be described in the example that 3:972-982 provides.
Coding can be obtained by gene engineering equally to the allelotrope of the insensitive phosphoglycerol dehydrogenase of Serine.
The PGD section of mediation Serine regulation and control is positioned at this proteic C-end region.Here it is why preferably at PGD PROTEIN C-terminal 25% section, import one or more amino acid whose insertions particularly preferably in terminal preceding 50 amino acid of C-, replaces or the reason of disappearance.These effects cause PGD that the susceptibility of Serine is reduced.
The allelotrope of this class of encoding PGDs can be by modifying serA gene 3 ' district, and the described PGD C-end region of promptly encoding realizes.In order to accomplish this point, Tu Bian serA gene is not recombinated on the cloning vector by recombinant DNA technology well known to those skilled in the art.This recombinant DNA process comprises restriction enzyme digestion, connect and conversion (ManiatisT., Fritsch E.F.and Sambrook, J., Molecular Cloning:A LaboratoryManual 1982, Cold Spring Harbor Laboratory).Special modification at structure gene 3 ' end can obtain by for example side-directed mutagenesis.
The example to the insensitive PGDs of Serine of expressing in the microorganism of the tryptophan metabolism that is adapted at regulating and control is listed in the table 1, and this table has shown their C-terminal amino acid sequence.Except the display part, this zymoprotein sequence and wild-type sequence are as broad as long.
Following measuring method is used to detect the active and Serine susceptibility of PGD of the allelic product of serA:
The PGD activity is by McKitrick, J.C. and LewisJ.p., and 1980, the method for introducing among the J.Bact.141:235-245 detects that the positive reaction of this enzyme and reversed reaction measure.Enzymic activity is not only measured under the Serine condition not having in this embodiment, also measures under the Serine condition of different concns.Said measuring method is fit to determine the Serine susceptibility of any phosphoglycerol dehydrogenase.Also can use other method to measure the PGD activity equally.
The mensuration that is used for this enzyme Serine susceptibility is the Ki value, that is to say, the concentration of Serine when suppressing this enzyme 50% activity.The Ki value and the C-terminal amino acid sequence of many anti-feedback serA allelotrope and wild-type serA gene (serAWT) are listed in the table 1.
Table 1: the allelic C-terminal amino acid sequence of sudden change serA
?Ber?AWT ?BerA?5 ?BerA?1508 ?BerA?11 ?BerA?1455 AEQG?V___?_NIA?AQYL?QTSA?QMGY?VVID?IEAD?EDVA?EKAL?QAMK?AIPG?TIRA?RLLY AEQG?V___?_NIA?AQYC?QTSA?QMGY?VVID?IEAD?EDVA?EKAL?QAMK?AIPG?TIRA?RLL_ AEQG?VCSR?ANIA?AQYC?QTSA?QMGY?VVID?IEAD?EDVA?EKAL?QAMK?AIPG?TIRA?RLLY AEQG?VCSR?ANIA?AQYC?QTSA?QMGY?VVID?IEAD?EDVA?EkAL?QAMK?AIPG?TIRA?RLL_ AEQG?V___?_NIA?AQYC?QTSA?QMGY?VVID?IEAD?EDVA?EKAL?QAMK?AIPG?TIR_?____ K 1/mM 0.02 0.2 3.8 50 100
Surprisingly press the present invention's allelic Ki value of production bacterial strain serA suitable and preferably preparation at 100 μ M and 50mM serine concentration.
Can use any recombinant vectors in bacterial strain of the present invention, expressing PGD albumen, thereby cause the allelic expression of the insensitive serA of Serine.A recombinant vectors that is fit to preparation bacterial strain of the present invention will have one to have the serA gene of the encoding serine susceptibility PGD lower than wild-type susceptibility and the carrier part that can duplicate automatically at least in F-strain.
The carrier example that can duplicate automatically in intestinal bacteria is listed in Pouwels P.H., Enger-Valk, B.E., Brammar W.J.1985, Cloning Vectors, Elsevier.Amsterdam.This class carrier comprises:
Plasmid with high copy number is as pBR322; PUC12,
Plasmid with medium copy number, as pACYC 184,177,
Plasmid with low copy number, as pSC 101,
Phage vector, as M13, the λ carrier.
Concerning a large amount of bacteriums, various carriers (for example the handful shape bacillus of EP0401735 and tyrothricin or CA 111 (1989) 168688q) have been described.
Suitable and preferred carrier is a carrier of waiting until low copy number in having; The carrier that has the p15A replicon, as pAYC 184 (ATCC 37033) or pACYC 177 (ATCC37031) for the most preferred.
A large amount of carriers for other bacterium be described in the literature (Pouwels et al., 1985, Cloning Vectors, Elsevier Sciencepublishers, Amsterdam).
Suitable recombinant vectors can produce by the standard technique of preparation recombinant DNA.These technology are described in detail in following document: as, Maniatis T., Fritsch E.F.andSambrookJ., 1982, Molerular Cloning:A Laboratory Manual, ColdSpring Harbor Laboratory, USA or Ausubel F.M.et al., 1987, Currentprotocols in Molecular Biology, Greenepublishing Associates, USA.
The preparation of recombinant vectors is, as, with restriction endonuclease donor bacterium DNA being digested to fragment earlier, this donor bacterium has the serA allelotrope of a coding to the insensitive PGD of Serine at karyomit(e) or on recombinant vectors.The fragment ordinary method of these cut-outs promptly is connected on the carrier molecule that digests with above-mentioned same restriction enzyme with the T4DNA ligase enzyme.After the ligation, the mixture currently known methods as calcium chloride shock or electroporation, transforms tryptophan metabolism and goes the recipient bacterium regulated and control.The carrier that contains needed serA gene can be in recipient bacterium, as, select or the complementation of serA sudden change obtains by aforesaid method such as antibiotics resistance.
Obtain bacterial strain of the present invention in addition-individual embodiment is that serA allelotrope is incorporated in the karyomit(e) as single copy, this on the one hand can suddenly change to induce with screening strategy and obtain by the above-mentioned initial strain that directly goes to regulate and control with tryptophane, on the other hand, also the serA allelotrope that is binned on the carrier can be incorporated on the karyomit(e) of producing bacterial strain, such integration method is well known.Can find the description of these technology in the document of following publication:
The integration of lambda particles phage mediation: Balakrishnan and Backmann, 1988, Gene67:97-103; Simons R.W.et al., 1987, Gene 53:85-89;
The gene that rec D-relies on is replaced: Shervell et al., 1987, J.Bact.141:235-245;
Other method: Silhavy et al., 1988, Experiments with GeneFusions, Cold Spring Harbor Laboratory.
The fermentation of bacterial strain of the present invention has disclosed astoundingly and has contained the anti-tryptophan metabolism that feed back serA allelotrope and go regulate and control of Serine Ki value between 100 μ M and 50mM, and containing the allelic bacterial strain of the trpE of tryptophane Ki value between 0.1mM and 20mM provides the highest tryptophane output.
The following examples are used to further specify the present invention.In the accompanying drawing:
Fig. 1 for the tryptophan metabolism approach and with the getting in touch and the gene of the main enzyme of encoding of serine metabolism.
Fig. 2 shows the position and the mutant trpE0 of △ trp L1 mutant (second class), trpE5, the position of trpE6 and trpE8 (first kind).
Fig. 3 is the nucleotide sequence of wild-type serA.
Fig. 4 is recombinant vectors pGC3.
Fig. 5 is the plasmid pGH5 after recombinating.
Fig. 6 is plasmid pKB1321.
Fig. 7 is plasmid pKB1508.
Fig. 8 is plasmid pGH11.
Fig. 9 is a plasmid pGH5/ II.
Figure 10 is a plasmid pGH11/ II.
Figure 11 is a plasmid pKB1508/ II.
Figure 12 is a plasmid pGH5/ III.
Figure 13 is a plasmid pGC3/ I.
Embodiment 1
Screening anti-feedback trpE allelotrope is also integrated these genes to karyomit(e).
The class jljl 5-methyl tryptophan of tryptophane is used to seek anti-feedback trpE allelotrope.N-methyl-N '-nitro-N-nitrosoguanidine (NG) is used as mutagenic compound.The initial strain that uses is e. coli k12 YMC9 ATCC 33927.The method of induced mutation based on the data of Miller (MillerJ.H., 1972, Experiments inMolecular Genetics.Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.:125-129).
Cultivation is in logarithmic phase in LB about 2 * 109 YMC9 cells and 50 μ g/ml NG 37C in 4ml 0.1M sodium citrate buffer solution (pH5.5) cultivated 30 minutes.After further using 0.1M (pH7.0) phosphate buffered saline buffer to wash 2 times, 0.1ml cell 37 ℃ of shaking culture in LB are spent the night.Then, with 0.9%NaCl the 0.1ml cell being made multiple concentration (10-3,10-4 and 10-5) is added on the minimum nutritional medium flat board that contains 100 μ g/ml 5-methyl tryptophans.Except that 5-methyl tryptophan, minimum nutrition base also contains: 5g/L glucose, 5mg/L VITMAIN B1,3g/L KH 2PO 4, 12g/L K 2HPO 4, 0.3g/LMgSO 4* 7H 2O, 0.1g/LNaCl, 5g/L (NH 4) 2SO 4, 14.7mg/L CaCl 2* 2H 2O, 2mg/LFeSO 4* 7H 2O, 1g/L trisodium citrate and 15g/L agar.At 37 ℃ after 24-48 hour, choose the 5-methyl tryptophan resistance clone, renewed vaccination is to flat board as stated above.
Mutants which had to above-mentioned acquisition is further determined the Ki value (Bauerle R.et al., 1987, Methods in Enzymology Vol.142:366-386) of trpE gene prod to tryptophane.It is possible mutant being divided into two classes in this way, and first kind mutant has anti-feedback o-amino benzoyl acid synthase, and the second class mutant has only the anthranilic acid synthase activity to improve and the constant enzyme of Ki value.
In order to determine at molecular level, the relevant DNA district of all kinds of mutant is cloned and is checked order, and for this reason, chromosomal DNA will separate under each situation and pair cut with Nhe I and two kinds of enzymes of Cla I.The fragment of isolating about 5kb size is connected to Nhe I/ClaIpBR322 size on the 4158bp plasmid fragment.Mixture after the ligation is transformed on intestinal bacteria KB862 (DSM7196) the trpE bacterial strain.Filter out the clone that can on the minimum nutritional medium of no tryptophane, grow.This complementary plasmid contains the Nhe I and the Cla I fragment of a 5kb size.Except that trpE and trpD are arranged, this 5kbNhe I/Cla I fragment also contain from the trpE upstream DNA district (about 0.8kb) and from the DNA district (about 1kb) in trpD downstream.Table 2 shows the aminoacid sequence difference and the Ki value of 4 first kind mutant.The sequence of mutant is identical with not marked other sequence on the wild-type sequence.
Table 2: the allelic aminoacid sequence of the trpE of sudden change
trpE?WT trpE?0 trpE?5 trpE?6 trpE?8 20 50 NPTA?LFHO?LCGD?RPAT?LLLE?SADI?DSKD?DLKS E S F E S E K 1/mM 0.01 0.1 3.0 15 ＞15
The sudden change of the second class mutant sequence analysis revealed both had been present in the manipulation subarea of trp promotor, was present in the DNA district of coding trp leading peptide again.The mutant that is called as Δ trpL1 and Δ trpL2 has 136bp and 110bp disappearance district respectively in the DNA district of coding leading peptide.Be numbered under AC V00372 number at the EMBL database, in the disappearance of Δ trpL1 mutant from the 33rd to 168 in Nucleotide, in the disappearance of Δ trpL2 mutant from the 11st to 120.
In order to obtain the stronger expression of anti-feedback trpE allelotrope, two class mutant are united.The second class mutant Δ trpL1 is used to this purpose.Fig. 2 shows the position and the mutant trpE0 of Δ trpL1 mutant (second class), trpE5, the position of trpE6 and trpE8 (first kind).
The 1.6kb Nru I fragment that contains Δ trpL1 mutant is separated from plasmid p Δ trpL (Fig. 2), with corresponding wild-type plasmid pE0, pE5, the Nru I fragment exchange of pE6 and pE8 (Fig. 2), the plasmid that obtains is hereinafter referred to as pIE0, pIE5, pIE6 and pIE8, and be used for chromosomal integration with the homologous recombination mode.For this reason, karyomit(e) Nhe I/Cla I fragment that size is about 5kb from each said plasmid is separated from low melting point agarose by embodiment 2 is described, and is transformed in the rec DpD106 bacterial strain [Δ trpLD102] with linear forms.The method for transformation that uses is the Calcium Chloride Method (Cohen et al., 1972, Proc.Natl.Acad.Sci.USA 69:2110-2114) of Cohen etc.Bacterial strain pD106 is preserved in Chinese typical culture collection center on September 11st, 1993, preserving number is CCTCC93047, and pressed budapest treaty, being deposited in German microbial preservation center (DSM) preserving number is DSM7195, can grow on the minimum nutritional medium of no tryptophane and to the penbritin sensitivity, promptly the bacterial strain of plasmid-free is screened comes out.The various anti-feedback trpE enzymes of encoding, and each trpE allelotrope that all is combined with Δ trpL1 mutant is transferred to KB862 (MillerJ.H. by the p1 transduction from relevant bacterial strain, 1972, Experiments in Molecular Genetics.Cold Spring Harbor, N.Y.:201-205).Bacterial strain KB862 has been preserved in Chinese typical culture collection center on September 11st, 1993, preserving number is CCTCC93048, and be preserved in German microbial preservation center (DSM) on July 28th, 1992 by budapest treaty, preserving number is DSM7196, filters out the bacterial strain of growing on the minimum nutritional medium of no tryptophane.These bacterial strains are called as pD103 (trpE0), KB862 (trpE5), SV164 (trpE8) and SV163 (trpE6).
Embodiment 2:
The preparation coding is to the serA gene of the insensitive phosphoglycerol dehydrogenase of Serine.
The serA wild type gene is cloned on the plasmid vector pUC18 from intestinal bacteria B strain (ATCC23226).
In order to obtain the chromosomal DNA of this bacterial strain, earlier with its overnight incubation in 37 ℃ of following Luria meat soups.Centrifugal then (4000g) collecting bacterial body cell, with (Ausubel et al., 1987,2.4.1-2.4.2 such as Ausubel, Currentprotocols inMolecular Biology, Greenepublishing Associates) this cell of method cracking and the purify DNA that provides.Measure the DNA amount that obtains at 260nm wavelength place with spectrophotometer.Output is about 600 μ g/100ml.
10 μ g chromosomal DNAs provide cutting of condition with Sph I Restriction Enzyme (Boehringer MannheimGmbH) by manufacturers.About 3 μ g fragment mixtures are connected to the plasmid vector pUC18 that can duplicate automatically with 0.2 μ g after the above-mentioned same enzyme Sph I cutting (being provided by Boehringer Mannheim GmbH) are provided, T4 ligase enzyme (being provided by Boehringer Mannheim GmbH) is provided the enzyme that uses, and reaction conditions is pressed manufacturer specification.Mixture after connecting is used to transform serA mutant pC1523 (CGSC#:5411; ) go up (CGSC:E.coli Genetic Stock Center, Department of Biology 255 OML, Yale University, Postbox 6666, New Haven, CT, USA).The method for transformation that uses is the Calcium Chloride Method (Cohen et al., 1972, Proc.Natl, Acad.Sci.USA 69:2110-2114) of Cohen etc.Bacterium after the conversion is coated on the minimum nutritional medium plate of no Serine.The clone that can grow under no Serine situation contains in Sph I fragment (about 3.5kb size) from colibacillary serA gene.The nucleotide sequence of wild-type serA gene is listed among Fig. 3, and the recombinant vectors that has the serA gene is called as pGC3 (Fig. 4).
SerA allelotrope serA5 can obtain by cutting plasmid pGC3 with restriction enzyme Sal I and Kpn I (being provided by Boehringer Mannheim GmbH) by the data of manufacturers.The fragment that obtains is separated with agarose electrophoresis, and size is 2.0kb, contains complete serA gene and comes out away from Sal I-Kpn I fragment purifying from glue of 8 codons of C-end.In order to accomplish this point, carry out electrophoresis so that only just can collect out DNA by melting agarose at low melting point agarose (providing) by BoehringerMannheim GmbH.Under the effect of T4 ligase enzyme (providing), be connected with the bifilar oligonucleotide that the pUC18 fragment of the Hind III/Sal I cutting of equimolar amount adds the preceding paragraph synthetic with this fragment 0.2 μ g by Boehringer Mannheim GmbH.The sequence of this section oligonucleotide is:
5′ 3′
C?ATT?CGC?GCC?CGT?CTG?CTG?TAA?TA
CTAGG?TAA?GCG?CGG?GCA?GAC?GAC?ATT?ATT?CGA 3′ 5’
This section oligonucleotide is 7 in last 8 codons of serA gene C-end, introduces terminated triplet TAA to replace the 8th codon.By the phosphoglycerol dehydrogenase of this serA genes encoding therefore by the brachymemma of a C-end amino acid top, this altered PGD aminoacid sequence is listed in (serA5) in the table 1.Plasmid after the reorganization is called as pGH5 (Fig. 5), transforms serA mutant pC1523 with connecting mixture.
SerA allelotrope serA1508 prepares as follows, by manufacturers's data, plasmid pGC3 cuts with Sph I/Sal I (being provided by Boehringer Mannheim GmbH) enzyme, the 3kb fragment that contains complete serA gene is by the gel electrophoresis purifying and be connected to on the pUC18 carrier that the Sph I/Sal I enzyme is cut, and the plasmid of acquisition is called pK1321 (Fig. 6).
PKB1321 acts on restriction endonuclease Hind II (being provided by BoehringerMannheim GmbH), and reaction conditions only allows partially digested (add the 0.05U enzyme among every μ gDNA, digested other reaction conditions by specification 10 minutes).This step especially is created in the fragment of being cut by the Hind II in the 1793rd of the serA gene.DNA joint with Xba I point of contact is inserted on this site by ligation.The sequence of DNA joint is as follows: 5 ' 3 ' TGC TCT AGA GCAACG AGA TCT CGT3 ' 5 '
This insertion will cause PGD to have 4 extra amino acid in this, and its sequence is listed in table 1.Have the plasmid that inserts section and be called pKB1508 (Fig. 7), and then be transformed among the serA mutant pC1523.
SerA allelotrope serA11 cuts the pGH5 plasmid with the reaction conditions that Sal I and Kpn I enzyme (being provided by Boehringer mannheim GmbH) provide by manufacturers by enzyme to obtain, and is purified into the fragment of 2.8kb size behind low melting point agarose gel electrophoresis from digestion mixture.This fragment contains from the carrier part of pUC18 with from the C-end region of serA5.Plasmid pKB1508 uses Sal I/Kpn I enzyme to cut too, is that the dna fragmentation of 2.0kb is separated from low melting point sepharose with size.This fragment contains the serA allelotrope serA1508 that band inserts mutant, but does not have 8 codons of C-end.Above-mentioned 2 fragments are connected together, be used to transform serA mutant pC1523, the recombinant plasmid of acquisition is called as pGH11 (Fig. 8).The phosphoglycerol dehydrogenase of this coding combines the insertion mutant serA5 deletion mutant of serA1508.The zone that has mutant in amino acid sequence coded is illustrated in table 1.
In order to produce the serA allelotrope that the bacterial strain expression suddenlys change, they are cloned on the carrier pACYC184 (ATCC37033), and this carrier is medium copy number.For accomplishing these,, isolate the dna fragmentation that each plasmid digestion back size is about 2kb and contains serA allelotrope serA5 and serA11 from low melting point sepharose with Sal I/Hind III digested plasmid pGH5 and plasmid pGH11.These two kinds of fragments are used for handling by product description from the Klenow fragment of the archaeal dna polymerase I of intestinal bacteria (being provided by Boehringer Mannheim GmbH) in different reaction solutions, its objective is that 5 ' end of the protrusion that digestion with restriction enzyme is cut becomes blunt end.For this reason, add among each 1 μ g of each fragment and contain 5U Klenow enzyme, 0.5mM dATP, dGTP, the 20 μ l reaction mixtures of dTTP and dCTP, the damping fluid of advising by manufacturers reacted 15 minutes for 37 ℃.
The dna fragmentation of each blunt end is connected on the pACYC184 carrier of cutting with pvu II enzyme, and this connects mixture and is used to transform serA mutant pC1523, and the plasmid after this augmenting is called as pGH5/ II and pGH11/ II (Fig. 9 and Figure 10) respectively.Plasmid pKBl508 cuts with Sal I/Sph I enzyme.To contain serA allelotrope serA1508 3.0kb fragment gel electrophoresis purifying.This fragment is connected with the pACYC184 that pvu II enzyme is cut then by the above-mentioned blunt end that is prepared into, and connects mixture and is transformed into intestinal bacteria pC1523, and this is called pKB1508/ II (Figure 11) by the plasmid after augmenting.
Plasmid pGH5/ II (serA5), pGH11 (serA11) and pKB1508 II are used to transform pD103 (trpE0), KB862 (trpE5), SV164 (trpE8) and SV163 (trpE6).
Embodiment 3
Make up the serA5 allelotrope of the anti-feedback of a chromosome coding with reorganization λ prophage.
For serA5 allelotrope being incorporated into karyomit(e) λ adsorption site (att λ), plasmid pRS551 (Simons et al., 1987, Gene 53:85-96) is arrived in this equipotential gene clone.For this reason, the Hind III/Sal I fragment (about 2kb) that will contain serA5 is separated from plasmid pGH5.5 ' protruding end is filled up with the Klenow fragment of archaeal dna polymerase I by product description, EcoR I joint is being connected back (Maniatis etal., 1982, Molecular Cloning:A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.:396-397), this 2kb fragment is connected on the carrier pRS551 that EcoR I enzyme cut.Recombinant plasmid is screened to come out and is called pRS5.
On plate, contain pRS5 rec A+ bacterial strain (as YMC 9 ATCC 33927) with λ RS45 phage splitting, use homologous recombination (Simons et al. again, 1987, Gene 53:85-96) an allos λ lysate that also contains the λ RS45 derivative of the serA5 that recombinates except that λ RS45 phage is produced in vivo.
SerApC1523 bacterial strain (CGSC#:5421) is used to screening reorganization λ RS45 derivative.For this reason, infect pC1523, be laid on then on the LB flat board that contains kantlex (25mg/L) with allos λ lysate (on seeing).The kalamycin resistance clone of the lysogeny that forms is examined their energy for growth on the minimum nutrition plate of no Serine.Filter out Serine-anauxotrophic clone and be used to prepare a strain homology serA5 λ lysate (adopting the ultraviolet induction method, Simons et al., 1987 Gene 53:85-96).
This homology serA5 λ lysate is used for infecting tryptophane and produces bacterial strain SV164.The SV164 att λ ∷ serA5 bacterial strain that obtains is by embodiment 4 described fermentations, and this special culture medium substitutes tsiklomitsin as selective agent with the 25mg/L kantlex in this case.
The output of tryptophane is about 12.5g/L, produces 3.5g/L with the identical bacterial strain that does not have serA5 and compares high.
Embodiment 4
Carry out tryptophane production with coryneform bacteria
Plasmid pGH5 cuts with restriction enzyme Sal I and Hind III (being provided by BoehringerMannheim GmbH) enzyme, and the big or small dna fragmentation that contains the serA5 gene for 2kb is separated from low melting point sepharose.The Klenow fragment (providing by Boehringer Mannheim GmbH) of e. coli dna polymerase I is provided again, this produced in fragments is become blunt end.Cut carrier pWST1 and be connected to the blunt end dna fragmentation with Sma I (providing) enzyme by Boehringer Mannheim GmbH.The pWST1 carrier is the carrier that can duplicate in intestinal bacteria and coryneform bacteria and can shuttle back and forth between this two bacterium, and the coryneform bacteria replicon of this carrier is from corynebacterium glutamicum ATCC19223 bacterial strain.The preparation of pWST1 carrier is at US-A4, is described in 965,197.Mixture after this ligation is used to transformed into escherichia coli pC1523 bacterial strain.Plasmid after augmenting is called pGH5/ III (Figure 12).
PGH5/ III plasmid is used to transform to produce tryptophane corynebacterium glutamicum ATCC21851, transforms to adopt electroporation technology to carry out, and this technology is by Wolf H.et al., and 1989, App1.Microbiol.Biotechnol.30:283-289 describes.The clone who contains recombinant plasmid pGH5/ III can screen containing on the 25mg/L kantlex agar plate by plasmid-encoded kalamycin resistance.
With restriction enzyme Sph I and Sal I digested plasmid pGC3.After containing the allelic 3kbDNA fragment purification of wild-type serA, be connected on the pWST1 carrier with above-mentioned method.The carrier pGC3/ I (Figure 13) that obtains is used to transform corynebacterium glutamicum ATCC21851.
The corynebacterium glutamicum ATCC21581 bacterial strain that contains serA allelotrope 1455 on a plasmid is by roughly the same method preparation.
Fermentation is disclosed in and contains the allelic bacterial strain of serA5 on the plasmid and can obtain high tryptophan output.
Embodiment 5
The influence that various plasmid-encoded serA allelotrope are produced various trpE bacterial strain tryptophanes.
(0.5%NaCl) the middle tsiklomitsin that adds makes into 15mg/l for 1% tryptone, 0.5% yeast extract, changes in the 100ml Erlenmeyer flask again, produces bacterial strains by in planting by the various tryptophanes of table 3 row at 10ml LB substratum.30 ℃ with the 150rpm rotational oscillation after 8-9 hour, and this pre-culture changes 100ml SM1 substratum.The SM1 substratum is by 5g/L glucose, 3g/L KH 2PO 4, 12g/L K 2HPO 4, 0.1g/L (NH 4) 2SO 4, 0.3g/L MgSO 4* 7H 2O, 15mg/L CaCl 2* 2H 2O, 2mg/L FeSO 4* 7H 2O, 1g/L trisodium citrate * 2H 2O, 1ml/L trace element solution (referring to down), 40mg/L L-phenylalanine, 40mg/L L-tyrosine, 5mg/L VITMAIN B1 and 15mg/L tsiklomitsin are formed.Trace element solution is composed as follows: 0.15g/L Na 2MoO 4* 2H 2O, 2.5g/L H 3BO 3, 0.7g/LCoCl 2* 6H 2O, 0.25g/L CuSO 4* 5H 2O, 1.6g/L MnCl 2* 4H 2O and 0.3g/L ZnSO 4* 4H 2O.Culture with 30 ℃ of 150rpm rotation concussions following 12-16 hour, is measured its OD afterwards in 1 liter Erlenmeyer flask 600Value is 2-4, and further fermentation is to carry out in the BIOSTATRM that is provided by Braun-Melsungen studies with fermentor tank.The cumulative volume of culture is 2 liters.
This substratum contains 17.5g/L glucose, 5g/L (NH 4) SO 4, 0.5g/LNaCl, 0.3g/L MgSO 4* 7H 2O, 15mg/L CaCl 2* 2H 2O, 75mg/LFeSO 4* 7H 2O, 1g/L trisodium citrate * 2H 2O, 1.5g/L KH 2PO 4, 1ml/L trace element solution (referring to last), 5mg/L VITMAIN B1 (VitB1), 0.75g/L-phenylalanine, 0.75g/L L one tyrosine, 2.5g/L yeast extract (Difco), 2.5g/L tryptone (Difco) and 20mg/L tsiklomitsin.
Glucose in the fermentor tank pumps into from 700g/L (W/V) glucose solution (autoclaving) with pump, makes that glucose concn reaches 17.5g/L in the fermented liquid, and before cultivation, adding tsiklomitsin in the fermentation, to make its final concentration be 20mg/L.In addition, with pump from 25%NH 4OH solution pumps into and makes pH remain on 6.7 in the jar.
The pre-culture of 100ml pumps in the fermenting container to be cultivated, and originally volume is about 1 liter, and culture begins to stir with 400rpm, is pressed into sterile air (flow velocity is 1.5vvm) with sterilization filter, and 30 ℃ ferment.
Use 25%NH 4OH keeps pH value 6.7 by automatic rectifier, in fermented liquid oxygen saturation during fermentation whenever should not be lower than 20%, the control of oxygen saturation is decided by stirring velocity when fermenting.
Every 2-3 hour, the glucose content of nutritive medium, optical density value and tryptophane output all should constantly be measured.Glucose content is that the glucose analyser enzymatic assays that is provided by YSI is provided.By continuous perfusion device glucose concn is remained between the 5-20g/L.Tryptophane in the substratum of fermentation back is measured with high performance liquid chromatography (HPLC).This culture a Nucleosil 100-7/C8 column chromatography for separation (250/4mm, Macherey-Nagel), this post carries out with the permanent solvent operation of flow velocity that 2ml/ divides, the moving phase of using is water/acetonitrile (83/17), adds 0.1ml H again in every liter 3PO 4(85%).Detect or use a diode-array detector, or with one 215 or the detection of 275nm fixed ultraviolet wavelength.In the time of 44-50 hour, ferment and stop.The tryptophane that produces in this fermentation after 48 hours is to calculate with g/L to list in the table 3.
Table 3: the tryptophane output of various serA/trpE combinations
SerAWT serA5 serA1508 serA11 serA1455trpE0 15.7 20.2 n.d. n.d. 6.7trpE5 12.5 18.9 15.0 20.0 7.5trpE6 11.6 24.1 13.8 24.0 4.0trpE8 7.5 18.0 n.d. 11.5 3.9n.d. undetermineds

Claims (8)

1, microorganism strains, it is characterized in that they have by the anti-tryptophan metabolism that feeds back trpE allelotrope and go to regulate and control and by the anti-serine metabolism that feeds back serA allelotrope and go to regulate and control, wherein the allelic tryptophane Ki of trpE value is 0.1mM-20mM, and the allelic Serine Ki of serA value is 0.1mM-50mM.
2, the bacterial strain of claim 1 is characterized in that serA allelotrope is incorporated in the karyomit(e).
3, claim 1 or 2 bacterial strain is characterized in that described microorganism is a bacterium.
4, the bacterial strain of claim 3 is characterized in that described bacterium belongs to bacillus coli.
5, the bacterial strain of claim 3 is characterized in that described bacterium belongs to Corynebacterium.
6, the method for preparation bacterial strain as claimed in claim 1 is characterized in that an anti-feedback serA allelotrope is introduced in the microorganism of the tryptophan metabolism that has.
7, the method for claim 6 is characterized in that described serA allelotrope is introduced in the karyomit(e) of microorganism of the tryptophan metabolism that has.
8, the application of each described bacterial strain of claim 1-5 in producing tryptophane.
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* Cited by examiner, † Cited by third party
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* Cited by examiner, † Cited by third party
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GB2304718B (en) * 1995-09-05 2000-01-19 Degussa The production of tryptophan by the bacterium escherichia coli
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US7300776B2 (en) * 2004-04-26 2007-11-27 Ajinomoto Co., Inc. L-amino acid-producing bacterium and a method for producing L-amino acid
RU2004137198A (en) * 2004-12-21 2006-06-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae Family In Which The yafA Gene Is Inactivated
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JP2010017081A (en) * 2006-10-10 2010-01-28 Ajinomoto Co Inc Method for producing l-amino acid
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RU2006143864A (en) 2006-12-12 2008-06-20 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE ENTEROBACTERIACEAE FAMILY IN WHICH THE EXPRESSION OF GENES cynT, cynS, cynX, OR cynR, OR THEIR COMBINATION IS DECREASED
JP2010041920A (en) 2006-12-19 2010-02-25 Ajinomoto Co Inc Method for producing l-amino acid
BRPI0806790B1 (en) 2007-01-22 2017-02-14 Ajinomoto Kk microorganism, and method for producing an 1-amino acid
JP2010088301A (en) 2007-02-01 2010-04-22 Ajinomoto Co Inc Method for production of l-amino acid
JP2010110216A (en) * 2007-02-20 2010-05-20 Ajinomoto Co Inc Method for producing l-amino acid or nucleic acid
JP2010110217A (en) 2007-02-22 2010-05-20 Ajinomoto Co Inc L-amino acid-producing microorganism and method for producing l-amino acid
DE102007051024A1 (en) 2007-03-05 2008-09-11 Evonik Degussa Gmbh Process for the preparation of L-amino acids using strains of the family Enterobacteriaceae
EP1975241A1 (en) 2007-03-29 2008-10-01 Evonik Degussa GmbH Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family
US8435769B2 (en) * 2007-04-13 2013-05-07 Monsanto Technology Llc Use of glyphosate to produce shikimic acid in microorganisms
WO2009031565A1 (en) 2007-09-04 2009-03-12 Ajinomoto Co., Inc. Amino acid-producing microorganism and method of producing amino acid
DE102007044134A1 (en) 2007-09-15 2009-03-19 Evonik Degussa Gmbh Process for the preparation of L-amino acids using improved strains of the family Enterobacteriaceae
RU2396336C2 (en) 2007-09-27 2010-08-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) METHOD OF PRODUCING AMINO ACIDS USING Escherichia GENUS BACTERIA
DE102007052270A1 (en) 2007-11-02 2009-05-07 Evonik Degussa Gmbh Process for the preparation of L-amino acids using improved strains of the family Enterobacteriaceae
EP2060636A1 (en) 2007-11-14 2009-05-20 Evonik Degussa GmbH Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family
JP2011067095A (en) 2008-01-10 2011-04-07 Ajinomoto Co Inc Method for producing target substance by fermentation process
KR20100120663A (en) 2008-01-23 2010-11-16 아지노모토 가부시키가이샤 Method of producing l-amino acid
RU2008105793A (en) 2008-02-19 2009-08-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО АГРИ) (RU) METHOD FOR DESIGNING OPERONS CONTAINING TRANSLATION-CONJUGATED GENES, BACTERIA CONTAINING SUCH OPERON, METHOD FOR PRODUCING USEFUL METABOLITIS AND METHOD FOR EXPRESS MONITORING
EP2246420B1 (en) 2008-02-21 2014-12-31 Ajinomoto Co., Inc. L-cysteine-producing bacterium, and method for production of l-cysteine
DE102008002309A1 (en) 2008-06-09 2009-12-10 Evonik Degussa Gmbh Process for the preparation of L-amino acids using improved strains of the family Enterobacteriaceae
DE102008040352A1 (en) 2008-07-11 2010-01-14 Evonik Degussa Gmbh Process for the preparation of L-tryptophan using improved strains of the family Enterobacteriaceae
DE102008044768A1 (en) 2008-08-28 2010-03-04 Evonik Degussa Gmbh Process for the preparation of organochemical compounds using improved strains of the family Enterobacteriaceae
JP5598329B2 (en) 2008-09-08 2014-10-01 味の素株式会社 Microorganism producing L-amino acid and method for producing L-amino acid
EP2382320B1 (en) 2009-01-23 2018-04-18 Ajinomoto Co., Inc. Process for producing l-amino acids employing bacteria of the enterobacteriacea family in a culture medium with controlled glycerol concentration
JP5359409B2 (en) 2009-03-12 2013-12-04 味の素株式会社 L-cysteine producing bacterium and method for producing L-cysteine
EP2460883A4 (en) 2009-07-29 2013-01-16 Ajinomoto Kk Method for producing l-amino acid
JP2012223092A (en) 2009-08-28 2012-11-15 Ajinomoto Co Inc Method for producing l-amino acid
RU2009136544A (en) 2009-10-05 2011-04-10 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) (RU) METHOD FOR PRODUCING L-CISTEINE USING THE ENTEROBACTERIACEAE FAMILY BACTERIA
JP2013013329A (en) 2009-11-06 2013-01-24 Ajinomoto Co Inc Method for producing l-amino acid
BR112012012915B1 (en) 2009-11-30 2020-12-01 Ajinomoto Co., Inc. method for producing l-cysteine, l-cystine, a derivative thereof, or a mixture thereof
RU2460793C2 (en) 2010-01-15 2012-09-10 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) Method for producing l-amino acids with use of bacteria of enterobacteriaceae family
JP2013074795A (en) 2010-02-08 2013-04-25 Ajinomoto Co Inc MUTANT rpsA GENE AND METHOD FOR PRODUCING L-AMINO ACID
RU2471868C2 (en) 2010-02-18 2013-01-10 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) Mutant adenylate cyclase, dna coding it, bacteria of enterobacteriaceae family containing said dan and method for preparing l-amino acids
RU2482188C2 (en) 2010-07-21 2013-05-20 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) METHOD FOR PREPARING L-ARGININE WITH USE OF BACTERIA OF GENUS Escherichia WHEREIN astCADBE OPERON IS INACTIVATED
RU2501858C2 (en) 2010-07-21 2013-12-20 Закрытое акционерное общество "Научно-исследовательский институт "Аджиномото-Генетика" (ЗАО АГРИ) METHOD FOR OBTAINING L-AMINOACID USING BACTERIUM OF Enterobacteriaceae FAMILY
JP5803927B2 (en) 2010-09-14 2015-11-04 味の素株式会社 Sulfur-containing amino acid-producing bacteria and method for producing sulfur-containing amino acids
JP2014036576A (en) 2010-12-10 2014-02-27 Ajinomoto Co Inc Method for producing l-amino acids
CN102140431B (en) * 2010-12-21 2014-08-27 大成生化科技(松原)有限公司 L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same
JP2014087259A (en) 2011-02-22 2014-05-15 Ajinomoto Co Inc L-cysteine-producing bacterium, and production method of l-cysteine
CN104160024A (en) 2011-04-01 2014-11-19 味之素株式会社 Method for producing l-cysteine
JP2014131487A (en) 2011-04-18 2014-07-17 Ajinomoto Co Inc Method for producing l-cysteine
RU2011134436A (en) 2011-08-18 2013-10-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") METHOD FOR PRODUCING L-AMINO ACID USING THE ENTEROBACTERIACEAE FAMILY POSSESSING AN INCREASED EXPRESSION OF GENES OF THE CASCADE OF THE FORMATION OF FLAGELLS AND CELL MOBILITY
CN102433312A (en) * 2011-11-30 2012-05-02 天津科技大学 D-3-phosphoglycerate-dehydrogenase as well as coding gene and construction method thereof
CN102453691B (en) * 2011-12-02 2013-09-18 山东鲁抗生物制造有限公司 Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan
HUE057864T2 (en) * 2012-01-10 2022-06-28 Cj Cheiljedang Corp Microorganisms of escherichia coli having enhanced l-tryptophan production and method for producing l-tryptophan using same
EP3312267B1 (en) 2012-01-30 2020-03-11 PTT Global Chemical Public Company Limited Production of muconic acid from genetically engineered microorganisms
EP2628792A1 (en) 2012-02-17 2013-08-21 Evonik Industries AG Cell with reduced ppGppase activity
US20150024440A1 (en) 2012-02-29 2015-01-22 Danmarks Tekniske Universitet Microorganisms for the production of melatonin
US20150037849A1 (en) 2012-02-29 2015-02-05 Danmarks Tekniske Universitet Microorganisms for the production of 5-hydroxytryptophan
EP2762571A1 (en) 2013-01-30 2014-08-06 Evonik Industries AG Microorganism and method for the production of amino acids by fermentation
RU2013118637A (en) 2013-04-23 2014-10-27 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE ENTEROBACTERIACEAE FAMILY IN WHICH THE yjjK GENE IS ADJUSTABLE
MY185322A (en) 2013-05-13 2021-05-04 Ajinomoto Kk Method for producing l-amino acid
JP2016165225A (en) 2013-07-09 2016-09-15 味の素株式会社 Method for producing useful substance
RU2013140115A (en) 2013-08-30 2015-03-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE EXPRESSION OF THE znuACB GENERAL CLUSTER IS DISORDERED
JP2016192903A (en) 2013-09-17 2016-11-17 味の素株式会社 Method for manufacturing l-amino acid from biomass derived from seaweed
EP3053999B1 (en) 2013-10-02 2019-11-20 Ajinomoto Co., Inc. Ammonia control apparatus and ammonia control method
RU2013144250A (en) 2013-10-02 2015-04-10 Закрытое акционерное общество "Научно-исследовательский институт Аджиномото-Генетика" (ЗАО "АГРИ") METHOD FOR PRODUCING L-AMINO ACIDS USING THE BACTERIA OF THE Enterobacteriaceae FAMILY IN WHICH THE EXPRESSION OF A GENE CODING A PHOSPHATE TRANSPORTER IS DECREASED
WO2015060314A1 (en) 2013-10-21 2015-04-30 味の素株式会社 Method for producing l-amino acid
BR112016008830B1 (en) 2013-10-23 2023-02-23 Ajinomoto Co., Inc METHOD FOR PRODUCING A TARGET SUBSTANCE
RU2014105547A (en) 2014-02-14 2015-08-20 Адзиномото Ко., Инк. METHOD FOR PRODUCING L-AMINO ACIDS USING THE ENTEROBACTERIACEAE FAMILY HAVING A SUPER EXPRESSED GENE yajL
RU2015120052A (en) 2015-05-28 2016-12-20 Аджиномото Ко., Инк. A method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family in which gshA gene expression is weakened
WO2017136795A1 (en) * 2016-02-04 2017-08-10 Synlogic, Inc. Bacteria engineered to treat diseases associated with tryptophan metabolism
WO2017146195A1 (en) 2016-02-25 2017-08-31 Ajinomoto Co., Inc. A method for producing l-amino acids using a bacterium of the family enterobacteriaceae overexpressing a gene encoding an iron exporter
EP3423563B1 (en) 2016-03-02 2023-05-24 PTT Global Chemical Public Company Limited Improved muconic acid production from genetically engineered microorganisms
JP7066977B2 (en) 2017-04-03 2022-05-16 味の素株式会社 Manufacturing method of L-amino acid
EP3385275B1 (en) 2017-04-07 2019-10-23 Evonik Degussa GmbH Method for producing aromatic l-amino acids using improved strains of the enterobacteriaceae family
US10858676B2 (en) 2017-05-22 2020-12-08 Ajinomoto Co., Inc. Method for producing objective substance
WO2019110101A1 (en) 2017-12-06 2019-06-13 Wacker Chemie Ag Strain of microorganisms and process for the fermentative production of methyl anthranilate
US11053526B2 (en) 2018-08-09 2021-07-06 Evonik Operations Gmbh Process for preparing L amino acids using improved strains of the enterobacteriaceae family
JP7094494B2 (en) * 2018-09-07 2022-07-04 味の素株式会社 Transformed plant of the genus Catharanthus in the family Apocynaceae
EP3861109A1 (en) 2018-10-05 2021-08-11 Ajinomoto Co., Inc. Method for producing target substance by bacterial fermentation
JP2022521544A (en) 2019-02-22 2022-04-08 味の素株式会社 Method for producing L-amino acid using a bacterium belonging to the family Enterobacteriaceae that overexpresses the ydiJ gene
WO2020204179A1 (en) 2019-04-05 2020-10-08 Ajinomoto Co., Inc. Method of producing l-amino acids
JP2022550084A (en) 2019-09-25 2022-11-30 味の素株式会社 Method for producing L-amino acid by bacterial fermentation
CN111926002B (en) * 2020-09-16 2021-01-05 中国科学院天津工业生物技术研究所 TrpE mutant and application thereof in gene engineering bacteria for producing L-tryptophan

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4371614A (en) * 1980-08-22 1983-02-01 Ajinomoto Co., Inc. E.Coli bacteria carrying recombinant plasmids and their use in the fermentative production of L-tryptophan
US4588687A (en) * 1981-11-24 1986-05-13 Ajinomoto Company Incorporated Method for producing L-tryptophan by fermentation
EP0293207A2 (en) * 1987-05-29 1988-11-30 The Standard Oil Company Eschericia coli carrying recombinant plasmid for the production of tryptophan
EP0338474A2 (en) * 1988-04-18 1989-10-25 Kyowa Hakko Kogyo Co., Ltd. Process for producing L-tryptophan
EP0401735A1 (en) * 1989-06-06 1990-12-12 Kyowa Hakko Kogyo Co., Ltd. Process for producing -L-tryptophan

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3385762A (en) 1964-06-29 1968-05-28 Chugai Pharmaceutical Co Ltd Process for the production of l-tryptophan by fermentation
FR2000641A1 (en) 1968-01-24 1969-09-12 Kyowa Hakko Kogyo Kk
JPS5119037B2 (en) 1972-11-16 1976-06-14
JPS575694A (en) 1980-06-10 1982-01-12 Showa Denko Kk Production of l-tryptophane
JPS5780398A (en) 1980-11-05 1982-05-19 Sanraku Inc Plasmid produced by genetic manipulation, coliform bacillus having the same and preparation of tryptophan with said bacillus
CA1226541A (en) 1984-01-13 1987-09-08 Stauffer Chemical Company Mutant strain deficient in l-serine deaminase activity
EP0232262A4 (en) * 1985-08-15 1989-09-19 Stauffer Chemical Co Tryptophan producing microorganism.
US4965191A (en) 1988-02-12 1990-10-23 Eastman Kodak Company Lower alcohol sulfate wash solution, test kit and method for the determination of an immunological ligand
DE69133587D1 (en) 1990-11-30 2008-01-03 Ajinomoto Kk Recombinant DNA sequences encoding enzymes free of feedback inhibition, plasmids containing these sequences, transformed microorganisms useful for production of aromatic amino acids, and their methods of preparation by fermentation
TW313589B (en) * 1991-12-12 1997-08-21 Wacker Chemie Gmbh

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4371614A (en) * 1980-08-22 1983-02-01 Ajinomoto Co., Inc. E.Coli bacteria carrying recombinant plasmids and their use in the fermentative production of L-tryptophan
US4588687A (en) * 1981-11-24 1986-05-13 Ajinomoto Company Incorporated Method for producing L-tryptophan by fermentation
EP0293207A2 (en) * 1987-05-29 1988-11-30 The Standard Oil Company Eschericia coli carrying recombinant plasmid for the production of tryptophan
EP0338474A2 (en) * 1988-04-18 1989-10-25 Kyowa Hakko Kogyo Co., Ltd. Process for producing L-tryptophan
EP0401735A1 (en) * 1989-06-06 1990-12-12 Kyowa Hakko Kogyo Co., Ltd. Process for producing -L-tryptophan

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101985638A (en) * 2010-12-01 2011-03-16 厦门大学 Method for producing L-tryptophan by precursor flow and fermentation
CN101985638B (en) * 2010-12-01 2013-04-17 厦门大学 Method for producing L-tryptophan by precursor flow and fermentation

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